tbuckley/cabot-search huggingface dataset:
Abstract P1155: P62 Overexpression Rescues Defective Autophagy Flux In Cardiomyocytes Deficient In P21 Activated Kinase 1
Authors: Shawn Geffken, Tint T Wun, Anoushka Guha, Pooja Jaiswal, Kinza Ahmed, Caleb Sooknanan, Tamayo Kobayashi, Satoru Kobayashi, Qiangrong Liang
Journal: Circulation Research (2023)

P21-Activated Kinase 1 (PAK1) is a serine-threonine kinase that plays a critical role in cardiomyocyte survival under numerous stressful conditions. However, the underlying mechanisms for PAK1’s cardioprotective effects remain unclear. Autophagy is a lysosome-dependent degradation pathway for eliminating and recycling long-lived proteins and unwanted cellular components, which is essential for cellular homeostasis. In this study, we investigated whether and how PAK1 regulates autophagy in cardiomyocytes. PAK1 was either silenced by siRNA knockdown or overexpressed by adenovirus-mediated gene transfer in H9c2 cardiac myoblast cells. Autophagy flux was determined by measuring LC3-II protein levels or by a fluorescent autophagy reporter with and without the lysosomal protease inhibitors pepstatin A (pepA) and Aloxistatin (E64d). PAK1 downregulation reduced LC3-II levels in the total cell lysates, which were not affected by pepA and E64d, suggesting that knockdown of PAK1 inhibited autophagy flux. Inversely, PAK1 overexpression increased LC3-II levels, which were further elevated by pepA and E64d, suggesting that PAK1 was sufficient to accelerate autophagy flux. Interestingly, the expression of p62, a ubiquitin-binding autophagic adaptor, was concurrently upregulated by PAK1 overexpression or downregulated by PAK1 knockdown, suggesting that p62 may mediates PAK1-dependent autophagy. Indeed, overexpression of p62 restored autophagy flux in the absence of PAK1 (Figure 1). Together, these results suggest that PAK1 is both sufficient and necessary for maintaining normal autophagy function in cardiomyocytes, which is likely mediated by p62.
Abstract P1022: Synthetic Copolymer-Based Membrane Stabilizers Confer Protection To Dystrophin-Deficient Striated Muscle <i>In Vitro</i> And <i>In Vivo</i>
Authors: Joseph D Quick, Dongwoo Hahn, John Bauer, Joseph Hassler, Brian Thompson, Benjamin Hackel, Tim Lodge, Frank Bates, Joseph M Metzger
Journal: Circulation Research (2023)

Duchenne muscular dystrophy (DMD) is a chronic and progressive muscle wasting disease resulting from the inability to produce the cytoskeletal protein dystrophin. The absence of dystrophin increases the susceptibility of the sarcolemma to mechanical damage and ultimately contributes to muscle degeneration and fibrosis. A growing body of evidence suggests that synthetic membrane stabilizing copolymers can be an effective strategy to limit contraction-induced sarcolemma damage. First-in-class copolymer-based membrane stabilizer Poloxamer 188 (P188) is a linear, amphiphilic triblock copolymer demonstrated to protect dystrophic cardiac and skeletal muscle in a wide variety of in vitro and in vivo models. However, the architectural features of P188 responsible for its membrane protective effects remain to be fully elucidated. Additionally, various other membrane-stabilizing copolymers differing in their size, architecture, and composition have been useful in structure-function studies. Here we sought to elucidate the properties of synthetic copolymers that confer stabilization to the dystrophin-deficient membrane. Using newly developed techniques, we designed and investigated an innovative class of branched “bottlebrush” copolymers at the single skeletal muscle fiber and organismal levels. Our results identify an intriguing, novel bottlebrush copolymer effective at previously unreported nanomolar concentrations in vitro . We also show in preliminary studies that bottlebrush copolymers may further confer protection during isoproterenol-induced cardiac stress testing in dystrophin deficient mice in vivo . These findings will be discussed in terms of copolymer-based membrane-interfacing molecules in the context of stabilizing dystrophin-deficient striated muscle.
Abstract P1183: Phosphorylation Signatures Of Human Heart Failure: Characterizing Dilated Cardiomyopathy-associated Signaling Pathways At The Cardiomyocyte Intercalated Disc
Authors: Cristine J Reitz, Marjan Tavassoli, Da Hye Kim, Saumya Shah, Robert Lakin, Allen Teng, Yu-Qing Zhou, Wenping Li, Sina Hadipour-Lakmehsari, Peter Backx, Andrew Emili, Gavin Y Oudit, Uros Kuzmanov, Anthony Gramolini
Journal: Circulation Research (2023)

Heart failure remains a highly prevalent condition with diverse etiology, yet the underlying signaling mechanisms are not fully understood. Despite the profound effects of post-translational protein modifications on downstream signaling, limited studies have investigated the cardiac phosphoproteome in human heart failure. We hypothesized that a combined proteomic and phosphoproteomic analysis of human dilated (DCM) and ischemic (ICM) cardiomyopathy would reveal novel etiology-associated disease pathways. Integrative analyses of left ventricular explants from DCM patients ( n =4) vs. non-failing controls ( n =4), and left ventricular infarct vs. non-infarct, and peri-infarct vs. non-infarct regions of ICM patients ( n =4) identified 5,570 unique proteins with 13,624 corresponding phosphorylation sites. Each pair-wise comparison revealed shared and etiology-specific signatures, with a unique DCM-associated enrichment of cell-cell adhesion pathways. We focused our attention on αT-catenin (CTNNA3) as a cardiomyocyte intercalated disc candidate phosphoprotein with a unique cluster of 4 hyperphosphorylated sites in DCM hearts ( P &lt;0.0001). Overexpression of non-phosphorylatable hCTNNA3 in ex vivo isolated adult mouse cardiomyocytes showed internalized protein expression and weaker cell-cell adhesion vs. wildtype (WT) and phospho-mimetic forms. We established an in vivo mouse model using recombinant adeno-associated virus 9 (rAAV9) harboring hCTNNA3-WT, hCTNNA3-phospho-null, or empty rAAV9 control. Phospho-null CTNNA3 mice developed left ventricular dilation and contractile dysfunction (% EF; 51.25±1.17 phospho-null vs. 62.07±1.20 WT vs. 66.76±1.42 empty; n ≥10) with impaired left ventricular conduction velocity (cm/s; 36.83±1.10 phospho-null vs. 47.74±2.04 WT; n =6), by echocardiography and ex vivo optical mapping. Loss of CTNNA3 phosphorylation led to intercalated disc remodeling with internalization and dissociation of CTNNA3, connexin 43, N-cadherin, β-catenin, and plakophilin 2 from the adherens junction, using high-resolution confocal imaging. Together these findings reveal a compensatory role for αT-catenin phosphorylation in maintaining cardiomyocyte intercalated disc organization in human DCM.
Abstract P3032: 15( <i>R</i> )-lipoxin A <sub>4</sub> Contributes To Splenic Erythrophagocytosis Via Activation Of ALX/FPR2
Authors: Brian Sansbury, Haley Asplund, Jing-Juan Zheng, Nolan Boyd, Jason Hellmann
Journal: Circulation Research (2023)

Red blood cells (RBCs) are the most abundant cell type in the body and are traditionally appreciated for their role in respiration and gas exchange. Recently, however, RBCs have garnered new attention for their immunomodulatory effects which is primarily achieved via their interaction with resident macrophages in the spleen, known as red pulp macrophages. As RBCs enter the spleen, red pulp macrophages probe the cells for signs of mechanical and oxidative damage, as well as inflammatory stimuli bound to cell surface receptors (e.g., foreign pathogens, endogenous chemokines). Once recognized, red pulp macrophages prune damaged portions of RBC membranes or phagocytose the cell entirely, efficiently processing and recycling the cellular cargo of the RBC. In a healthy human, this process, termed erythrophagocytosis, accounts for the daily turnover of roughly 150-200 billion RBCs. Consequently, defects in the system can have wide-ranging health effects including anemia, impaired host defense, and even sepsis. Recent data suggest that arachidonic acid (AA)-derived prostaglandins promote erythrophagocytosis, yet the impact of other eicosanoid species on the process has not been well described. We performed targeted lipidomics analyses focused on the AA metabolome in two distinct models of erythrophagocytosis. First, mouse bone marrow-derived macrophages were incubated with freshly isolated RBCs; then, spleens from mice were collected following exogenous delivery of damaged RBCs. In both models, we found increased production of the 15( R )-epimer of lipoxin A 4 (15( R )-LXA 4 ), as well as its biosynthetic intermediate, 15-HETE. Similar to LXA 4 , 15( R )-LXA 4 has been shown to robustly promote the active resolution of inflammation by regulating leukocyte function via its interaction with the G protein-coupled receptor, ALX/FPR2. In mice genetically deficient in this receptor, we measured a significant decrease in splenic clearance of damaged RBCs. Together, these data suggest that 15( R )-LXA 4 activation of ALX/FPR2 on red pulp macrophages may be a critical aspect regulating erythrophagocytosis and maintaining RBC homeostasis.
Abstract P2029: Ror2-mediated Proteostatic Disruption As A Novel Mechanism For Right Ventricular Failure
Authors: Jonathan J Edwards, Gen Uy, Keita Uchida, Emily Scarborough, Yifan Yang, Jeff Brandimarto, Li Li, Nora Yucel, Benjamin Prosser, Zoltan Arany
Journal: Circulation Research (2023)

No therapies exist to reverse right ventricular failure (RVF), in part because RVF molecular drivers have been incompletely characterized. We recently identified RVF-severity dependent re-expression of the developmental noncanonical WNT receptor Ror2 in dilated and ischemic cardiomyopathy RV. We now demonstrate that Ror2 plays a novel role in regulating cardiomyocyte proteostasis (Figure). NRVMs with Ror2 overexpression (Ror2 OE ) are smaller, rounder and demonstrate fragmented sarcomeres, loss of intercalated disc proteins, and reduced ubiquitination. Knockdown of Ror2 (Ror2 KD ) reveals opposing phenotypes. RNAseq of Ror2 OE vs Ror2 KD revealed striking downregulation of chaperone genes (Hspa1a/b, Cryab, Hsp90aa1, Hsph1/d1, etc.) and upregulation of genes involved in proteolysis (Rcn3, Nkd2, Mapk12). Using Suc-LLVY-AMC and GFP-degron, we find that proteasome activity is enhanced in Ror2 OE and inhibited in Ror2 KD . MG-132 proteasomal inhibition reversed structural phenotypes in Ror2 OE NRVMs. In vivo cardiac Ror2 OE caused RV dilation and systolic and diastolic dysfunction, more prominently in male mice. In male Ror2 OE RVs, proteasome activity was enhanced and ubiquitin staining of RV cardiomyocytes was reduced. Finally, PAB caused a 5-fold increase in triton soluble and 40-fold increase in triton insoluble Ror2 expression, which correlated to severity of RV hypertrophy and dilation. Studies to test the efficacy of Ror2 KD for PAB RVF are ongoing. In sum, our work suggests that reactivation on non-canonical Wnt signaling contributes to RVF, unveiling a novel potential therapeutic target.
Abstract P1131: Epicardially-secreted Fibronectin Promotes Hesc-cardiomyocytes' Maturation In 3d-engineered Heart Tissues.
Authors: Lay Ping Ong, Johannes Bargehr, Vincent Knight-Schrijver, Jonathan Lee, Semih Bayraktar, Maria Colzani, Silvia Marchiano, Alessandro Bertero, Charles Murry, Laure Gambardella, Sanjay Sinha
Journal: Circulation Research (2023)

Ischaemic heart failure is due to the irreversible loss of cardiomyocytes. Preclinical studies showed that human pluripotent stem cell (hPSC)-derived cardiomyocytes could regenerate infarcted hearts and improve cardiac function. However, these hPSC-derived cardiomyocytes remained immature. Epicardial-myocardial crosstalk underpins key events during cardiac development and maturation. Recently we showed that incorporating hPSC-derived epicardial cells improved cardiomyocyte maturation in 3D-engineered heart tissues (3D-EHTs). However, the key signaling pathways underpinning epicardial-myocardial crosstalk remain unclear.We found that Fibronectin (FN1) was uniquely upregulated in 3D-EHTs co-cultured with hESC-epicardium, compared to other stromal cells such as mesenchymal stem cells. Thus, we posited that epicardially-secreted fibronectin (FN1) is a key mediator of epicardial-cardiomyocyte crosstalk driving cardiomyocyte maturation.To test this hypothesis, we performed a series of loss of FN function experiments with peptide inhibition (pUR4), CRISPR/Cas9-mediated FN1 knockout, or tetracycline-inducible FN1 knockdown in 3D-EHTs. We found that the loss of epicardial-FN, at both protein and gene levels, resulted in immature hPSC-derived cardiomyocytes with decreased contractile function, inefficient Ca2+ handling, and disorganized sarcomeric apparatus. Conversely, when we supplemented 3D-EHTs with recombinant human FN1, we could recover hPSC-derived cardiomyocyte maturation. Furthermore, our RNA-sequencing analyses found FN1 within a wider paracrine network of epicardial-cardiomyocyte crosstalk, thus solidifying FN1 as a key driver of hPSC-derived cardiomyocyte maturation in 3D-EHTs.
Abstract P1129: Directing Revascularization In Vivo With Local Controlled Release Of An Optimized Angiogenic Growth Factor Cocktail
Authors: Alicia Minor, Stephanie Roser, Rajeev Kant, Keyana Zahiri, Collin Polucha, Kareen L Coulombe
Journal: Circulation Research (2023)

Reduced vascular perfusion due to aging, ischemia, or injury impedes healing and tissue function in conditions like skin ulcers and myocardial infarction, motivating development of therapies to restore vasculature locally in target tissues. We hypothesize that specific combinations of signaling molecules stimulate angiogenesis and vascular remodeling in the local tissue environment to increase perfused vessel density. Using a vascular tissue engineering approach with a fractional factorial design of experiments (DOE), we evaluated in vivo angiogenesis of combinations of ten growth factors, cytokines and/or enzymes including VEGF, bFGF, Shh, PDGF, IGF-1, EGF, TGF-β1, Ang-1, MCP-1 and MMP-9 in a subcutaneous gel plug assay in the WT immune competent rat. Our initial assessment with Matrigel identified 5 factors that maximized CD31+ vessel lumen density and aSMA+ encoated vessels at 7 days, warranting further evaluation. We developed a fully defined biomaterial system with covalently bound heparin to alginate mixed with collagen type 1 as an injectable gel. This defined biomaterial prolonged protein release up to 14 days in vitro versus Matrigel and unmodified alginate (2-4 days). The defined biomaterial was used for a second round DOE to deliver high (2 μg) and low (0.5 μg) doses of VEGF, bFGF, Shh, PDGF, and/or IGF-1. The defined biomaterial alone enhanced average lumen diameter 15-fold and endothelial cell infiltration 12-fold versus Matrigel. Perfused lumen density increased 16-fold with high Shh; aSMA-positive lumen wall area increased 4-fold with high VEGF, PDGF, and IGF-1; perfused vessel size increased 3-fold with high VEGF, bFGF, and Shh; and endothelial cell recruitment increased 2-fold with high IGF-1 relative to unloaded controls after 7 days in vivo. Empirical testing of 2- to 5-factor combinations showed that cocktail potency required initiation of angiogenic sprouting by VEGF or bFGF and vascularization responses were strongest in the presence of Shh and IGF-1. In conclusion, we identified sets of highly potent cocktails and leveraged controlled release from a defined biomaterial as a tailored revascularization therapy for local angiogenesis, vascular remodeling, and tissue regeneration.
Abstract P1102: Organic Semiconductor Photomodulation Enhances Maturation Of Pluripotent Stem Cell-derived Cardiomyocytes
Authors: Camilla Galli, Nicolo Salvarani, Carlotta Ronchi, Gabriele Tullii, Camilla Marzuoli, Marco Malferrari, Stefania Rapino, Maria Rosa Antognazza, Elisa Di Pasquale
Journal: Circulation Research (2023)

Cardiomyocytes differentiated from pluripotent stem cells (PSC-CMs) hold a great potential for the study and the cure of cardiovascular disease; indeed they have been largely used as platform for disease modeling and drugs testing, and represent a promising source of cells for regenerative therapies. However, their immature phenotype represents a major hurdle for their full application; indeed, PSC-CMs differ from adult ones for molecular, metabolic and morpho-functional properties. Recently, a new technology based on optical excitation of organic semiconductors (OS) has been shown to be able to modulate the behavior of many cell types by targeting different cellular processes, as proliferation, angiogenesis and calcium signalling. Here, we adopted a multidisciplinary approach based on morpho-functional, metabolic and transcriptional analyses to study the effect of OS-photoexcitation on PSC-CMs, by the use of a red-light sensitive OS polymer, namely Poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta [2,1-b;3,4-b']dithiophene)-alt-4,7(2,1,3-benzothiadiazole)] (PCPDTBT), in the form of a thin film. Our results revealed a significant modulation of markers of maturation in PSC-CMs exposed to PCPDTBT and photomodulation, showing increased size, a shift of their maximal diastolic potential toward more negative values, and augmented Ca 2+ transient amplitude. Moreover, by Scanning ElectroChemical Microscopy (SECM) we found a decrease in glucose uptake and lactate release upon PCPDTBT light stimulation, potentially indicating a switch toward a more adult-like metabolism in stimulated PSC-CMs. Recently obtained RNA sequencing data are in line with these results, and will provide us with hints on the underlying molecular mechanisms. In conclusion, although not definitive, our data are in support of a potential effect of polymer-mediated optical photoexcitation in boosting PSC-CMs toward a more mature phenotype. The demonstration of a relevant effect of OS-photomodulation on PSC-CM maturation and functionality will significantly promote their full applications toward personalized medicine.This research was supported by EU Horizon 2020 FETOPEN- 2018-2020 Programme ‘LION- HEARTED’-GA #828984
Abstract P1119: Deep Learning-based Cardiac Microphysiological Systems For Studying Reentry Arrhythmia
Authors: Young Hoon Son, Hyeong Kyun Park, Christina Sheng, Jeongin Jang, Junbeom Park, Sung-Jin Park
Journal: Circulation Research (2023)

Arrhythmias can be caused by reentry and focal activities, leading to a disruption of the normal sinus rhythm. Understanding these mechanisms and their contributions to arrhythmias in individual patients is crucial for developing effective treatments. To achieve this, in vitro human models based on induced pluripotent stem cell-derived cardiomyocytes have been developed to replicate arrhythmogenic activities and identify pathophysiological mechanisms specific to patients. However, the current methods for analyzing electrophysiology (EP) videos are limited and subjective, making the interpretation of clinical EP videos challenging. To address these limitations, we have developed a deep cardiac EP system called cEP-Net. The system comprises a cardiac microphysiological system designed for reproducing various forms of reentry and focal activities in vitro and a deep learning-based EP video classifier with a spatial and temporal streams architecture. The deep EP classifier analyzes cardiac EP videos, identifies arrhythmogenic activities, and categorizes videos into four categories based on their characteristics: (1) localized activities, (2) normal linear propagation, (3) focal activity, and (4) reentrant activity. We pre-trained cEP-Net using 111,110 annotated in vitro EP videos produced by a cardiac microphysiological system and evaluated its performance for in vitro and in vivo clinical settings. Our pre-trained cEP-Net demonstrated high Kappa (91.0%) and concordance rate (94.7%) for all categories. cEP-Net visualized in vitro EP videos in UMAP domains and successfully clustered them into four categories. Additionally, cEP-Net successfully captured the development and evolution of reentry during early in vitro cardiac tissue formation and the initiation of focal activities in the HCN4 (a pacemaker protein)-expressing tissues. We also used cEP-Net to quantify the arrhythmogenic risks of cardiac agents and found that cEP-Net could capture Flecainide-induced vulnerability to reentry. Lastly, we applied cEP-Net to analyze electroanatomical mapping videos in vivo. The cEP-Net demonstrated its capability to identify phase singularities in hearts with atrial fibrillation (AF) and capture the EP patterns of persistent AF hearts.
Abstract P2008: Contractile Adaptation Of The Right Ventricular Myocardium In Pulmonary Hypertension
Authors: Rana Raza R Mehdi, Sunder Neelakantan, Eric Wang, Peng Zhang, Gaurav Choudhary, Reza Avazmohammadi
Journal: Circulation Research (2023)

Introduction: Pulmonary hypertension (PH) is characterized by increased right ventricular (RV) afterload inducing notable biomechanical remodeling in the RV free wall (RVFW) myocardium. While changes in the passive behavior of the RVFW have been studied, its contractile adaptations in PH remain to be investigated. Methods: Two sugen-hypoxia models were developed by injecting SU5416 into Sprague Dawley (SD) and Fischer (CDF) rats (n=4 each) and placing them in a hypoxia chamber for 3 weeks, followed by 1 week of normoxia. Wild-type rats (n=4), injected with vehicle and kept in normoxia, were used as controls. Right heart catheterization was perfomed at week 4. RVFW samples were skinned in 10% Triton. Samples were activated at pCa 4.5. Mechanical tests were performed isometrically at 20% stretch. Circumferential and longitudinal stresses were summed to report ensemble stress. Results: RV systolic pressure and stroke volume showed significant increase and decrease in both PH groups, respectively, compared to controls (Figs. 1A, B). While increases in initial stress, representing the passive behavior, did not reach significance in either PH group (Fig. 1C), total stress, representing the combined passive and active behavior, increased significantly only in CDF-PH group (Fig. 1D), known to exhibit a more maladaptive RV remodeling compared to SD-PH. Select stresses are shown in Figs. 1E, F. Conclusions: While elevated passive stresses in the RVFW are commonly reported in PH, the measurement of contractile stresses is essential to properly assess the adaptation of the RV. Further studies are needed to determine the role of passive vs. active stresses in the outcome of RV remodeling.